1. Community Behavior and Spatial Regulation within a Bacterial Microcolony in Deep Tissue Sites Serves to Protect against Host Attack 
Kimberly M. Davis, Sina Mohammadi, Ralph R. Isberg 
Cell Host & Microbe 
Volume 17, Issue 1, Pages (January 2015)
DOI: /j.chom
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2. Cell Host & Microbe 2015 17, 21-31DOI: (10.1016/j.chom.2014.11.008)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1
Spatial Control of Y. pseudotuberculosis Gene Expression during Growth in Tissues
C57BL/6 mice were inoculated intravenously with 103 WT Y. pseudotuberculosis, and spleens were harvested at day 3 postinoculation (PI).
(A) Inoculated with an equal mixture of Yptb GFP+ (Ptet::gfp expressed from pACYC184, lacking tetO) and Yptb mCherry+ (yopE::mCherry, integrated downstream of yopE+). Sections stained with Hoechst and visualized by fluorescence microscopy. Seven mice were analyzed, with three to 11 centers/mouse.
(B) Three distinct microcolonies (arrows), low-power magnification.
(C) Low- and high-power magnification of hematoxylin and eosin (H&E)-stained sections from a mouse infected with WT, centered on area of inflammation (dashed box, I). R, red pulp region; I, area of inflammation.
(D) Bacterial transcripts quantified by quantitative real-time PCR. Values expressed relative to 16S. Each bar represents an individual mouse (numbered, black bars) or the inoculum (white bars, error bars depict median and range).
(E) Nos2 transcripts quantitified by quantitative real-time PCR, relative to Gapdh, and relative to uninfected controls. Each bar represents an individual mouse (numbered), and corresponds to the mice in (D).
(F) Mice inoculated with the WT gfp+ Phmp::mCherry strain. Frozen splenic sections stained with Hoechst to detect host nucleic acids. Ratiometric image of the mCherry (Phmp) signal/GFP signal (range, 0.2–2) displayed using rainbow scale (Experimental Procedures).
See also Figure S1.
Cell Host & Microbe  , 21-31DOI: ( /j.chom )
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4. Figure 2 Hmp Activity Is Required for Spatial Regulation of Phmp
C57BL/6 mice intravenously inoculated with WT gfp+ Phmp::mCherry or Δhmp gfp+ Phmp::mCherry strains and spleens harvested at day 3 PI. Frozen sections were prepared, and bacterial reporter expression was visualized by fluorescence microscopy.
(A) Size-matched representative images of four WT and four Δhmp microcolonies.
(B) Colorimetric depiction of signal intensity (scale bars) in each channel (top, mCherry; bottom, GFP) from the bottom images in (A).
(C) hmp(mCherry)/GFP ratios generated by dividing signal intensity of each channel at the centroid and periphery of each replication center. Each dot represents an individual microcolony, two to 12 centers analyzed/mouse, five mice. The dotted line: signal intensity equivalent for each channel.
(D) Δhmp rescued by integrating hmp::mCherry construct into chromosome. Representative microcolony shown alongside hmp(mCherry)/GFP ratios, generated as described in (C). One to ten centers analyzed/mouse, five mice.
Statistics: Mann-Whitney (comparison between centroids), Wilcoxon-matched pairs (comparison between centroid and periphery), ∗∗∗p < 0.001, n.s., not significant. Scale bars, 20 μm. See also Figure S2.
Cell Host & Microbe  , 21-31DOI: ( /j.chom )
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5. Figure 3
Distinct Subpopulations of Peripheral Bacteria within Microcolonies
(A) Increasing magnifications of area of inflammation (I, dashed box) from splenic section, stained with H&E. W, white pulp region; R, red pulp region; I, area of inflammation.
(B) C57BL/6 mice intravenously inoculated with the WT gfp+ yopE::mCherry or Δhmp gfp+ yopE::mCherry strains and spleens harvested at day 3 PI. Frozen sections prepared and visualized by fluorescence microscopy.
(C) yopE(mCherry)/GFP ratios generated as described in Figure 2C. One to 11 centers analyzed/mouse, five mice/infection.
(D) C57BL/6 mice intravenously inoculated with WT Phmp::gfp yopE::mCherry or Δhmp Phmp::gfp yopE::mCherry strains and spleens harvested at day 3 PI. Frozen sections were prepared and visualized by fluorescence microscopy. Representative images are shown with colorimetric depiction of signal intensity for each channel.
(E) hmp(GFP)/yopE(mCherry) ratios generated as described in Figure 2C. Two of 11 centers analyzed/mouse, six mice/infection.
(F) Nitrogen stress genes induced with NO2 (NO2, +), and/or type III secretion system genes induced by low Ca2+ conditions (T3SS, +), in cultures of the WT strain. Transcription levels expressed relative to 16S. Each bar represents four replicates; error bars depict the median and range.
Statistics: Mann-Whitney (in vitro transcription), Wilcoxon matched pairs (for comparison between centroid and periphery), ∗∗∗p < 0.001, ∗∗p < 0.01, ∗p < 0.05, n.s., not significant.
Cell Host & Microbe  , 21-31DOI: ( /j.chom )
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6. Figure 4 Bacteria Do Not Directly Contact iNOS-Producing Host Cells
(A) Nos2−/− mice infected with WT gfp+ Phmp::mCherry strain at day 3 PI. Spleen frozen sections were prepared, stained with antibodies for indicated host markers, and visualized by fluorescence microscopy. Twenty-three centers were analyzed across two mice.
(B) C57BL/6 mice intravenously inoculated with WT (black bars, five mice) or PBS (white bars, three mice); error bars depict median and range. Splenocytes were detected by flow cytometry. Values represent percentage of each cell type within total population of iNOS+ cells.
(C) Splenocytes stained and detected by flow cytometry. Values represent percentage of iNOS+ cells within each cell type.
(D) Low- and high-power magnification of an area of inflammation (dashed box, I), from splenic section of a mouse infected with WT. W, white pulp region; R, red pulp region; I, area of inflammation.
(E and F) C57BL/6 mice intravenously inoculated with GFP+ WT strain, splenic frozen sections prepared at day 3 PI and stained with antibodies. (E) Low-power magnification. (F) High-power magnification. Inset: zoom of colony interface.
(G) C57BL/6 mice intravenously inoculated with GFP+ WT, splenic sections prepared at day 3 PI, and antibody stained. Nine representative images are shown.
(A) and (E)–(G) all depict distinct microcolonies; 115 microcolonies were analyzed from 14 mice. See also Figure S3.
Cell Host & Microbe  , 21-31DOI: ( /j.chom )
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7. Figure 5 The Absence of hmp Causes Reduced Fitness
(A) Balb/c mice intravenously inoculated with the GFP+ WT or GFP+ Δhmp strain and spleens harvested at days 3 and 5 PI. Microcolony areas (μm2) were quantified (Experimental Procedures) in five mice, with six to ten total centers/mouse.
(B) WT and Δhmp microcolonies at day 5 PI.
(C) C57BL/6 mice intravenously inoculated with an equal mixture of mCherry+ (yopE::mCherry) WT and GFP+ Δhmp strains, and spleens harvested at day 3 PI. Competitive index: ratio of Δhmp/WT spleen colony-forming units at day 3, divided by the ratio of Δhmp/WT colony-forming units in inoculum. Each dot, individual mouse. Dotted line, each strain has equal fitness. WT and mutant microcolony areas (μm2), quantified (Experimental Procedures) in seven mice, with six to ten total centers/mouse.
(D) Representative WT and Δhmp microcolonies from same organ shown at day 3 PI.
Statistics: Mann-Whitney, ∗∗∗p < 0.001, ∗∗p < Scale bars, 50 μm. See also Figure S4.
Cell Host & Microbe  , 21-31DOI: ( /j.chom )
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8. Figure 6
Bacteria within Microcolonies Fail to Activate Postexponential Genes
(A) C57BL/6 mice intravenously inoculated with WT strain and spleens harvested at day 3 PI. Bacterial RNA was isolated and reverse transcribed, and transcripts were detected by quantitative real-time PCR. Transcription levels expressed relative to 16S. Bars, individual mice or mean of culture condition (six replicates/culture condition).
(B) C57BL/6 mice intravenously inoculated with WT PkatG::gfp yopE::mCherry strain, and spleens harvested at day 3 PI. Frozen sections prepared and visualized by fluorescence microscopy. Two to eight centers analyzed/mouse, five mice. Three representative images are shown.
Scale bars, 20 μm. See also Figure S5.
Cell Host & Microbe  , 21-31DOI: ( /j.chom )
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